Isolation and Characterization of Microsatellite Loci in the Chinese Cobra Naja atra (Elapidae)

Long-Hui Lin,Lu-Xi Mao,Xiang Ji,Yan-Fu Qu,Xia Luo

doi:10.3390/ijms12074435

Abstract

We characterize thirteen polymorphic microsatellite loci isolated from Naja atra genomic libraries, which were enriched for AC-motif microsatellites. The thirteen loci were screened on a group of 48 individuals from two populations, one in Yong’an and the other in Ganzhou. These markers revealed a relatively high degree of genetic diversity (4–12 alleles per locus) and heterozygosity (Ho ranged from 0.213–0.854 and He ranged from 0.301–0.838). Tests for departure from Hardy-Weinberg equilibrium and for linkage disequilibrium were conducted for each of the two populations separately. After sequential Bonferroni correction, none of the 13 loci showed significant departures from Hardy-Weinberg equilibrium. Hierarchical analysis of molecular variance indicated that a small but significant (P < 0.001) proportion (16.0%) of the total variation in the microsatellite DNA data were attributable to differences among populations, indicating geographical structuring and restricted gene flow. It could be attributable to the Wuyi mountains in the area having a sufficiently isolating effect to significantly reduce gene flow. Our microsatellite data also showed a low Nm (1.31) value in the two populations from mainland China. Thus, the Yong’an and Ganzhou populations could be treated as distinct evolutionarily significant units (ESUs). The high level of polymorphism revealed by these microsatellite markers will be useful for the study of gene flow, population structure and evolutionary history of N. atra.

Highlights

The Chinese cobra (Naja atra) is an oviparous elapid snake with a distribution covering southeasternChina and northern Vietnam [1]
Genomic DNA was used to construct a library enriched for AC-motif microsatellite sequences
All PCRs were performed with the following conditions: 100–200 ng of genomic DNA, 10 μL 2 × EasyTaq PCR SuperMix polymerase (TransGen Biotech), 0.5 μM of each primer; 5 min denaturation at 95 C; 32 cycles of 30 s at 95 C, 1 min at specific annealing temperatures, and 1 min at 72 C (Table 1); and a final extension of 72 C

Summary

Introduction

The Chinese cobra (Naja atra) is an oviparous elapid snake with a distribution covering southeasternChina (including Taiwan, Hongkong and Hainan) and northern Vietnam (see Figure 1) [1]. To develop effective conservation strategies for the cobra, we need a better understanding of gene flow, population structure and evolutionary history of this species. We describe the isolation and characterization of thirteen microsatellite markers in N. atra. Genomic DNA was used to construct a library enriched for AC-motif microsatellite sequences.

Results

Conclusion